Method of preventing decrease in the activity of protein due to freezing and use of polypeptide in the production of protein activity decrease preventing agent

ABSTRACT

An objective of the present invention is to provide a means to prevent freeze-induced decrease in protein activity. The present invention relates to a method for preventing freeze-induced decrease in protein activity comprising adding a polypeptide comprising the following amino acid sequence (I) to a protein to be frozen. Further, the present invention relates to use of a polypeptide comprising the following amino acid sequence (I) to produce an agent for preventing freeze-induced decrease in protein activity:  
                     (SEQ ID NO: 1)                   Ser-Ser-Thr-Gly-Ser-X1-Ser-X2-Thr-Asp-X3-X4-X5-X6-               X7-X8-Gly-Ser-X9-Thr-Ser-Gly-Gly-Ser-Ser-Thr-Tyr-           Gly-Tyr-Ser-Ser-X10-X11-X12-X13-Gly-X14-Val (I).

[BACKGROUND OF THE INVENTION]

[0001] 1. Field of the Invention

[0002] The present invention relates to a method for protecting proteinsfrom denaturation which occurs when the proteins are frozen. Further,the present invention is relates to use of polypeptides for protectingproteins from such denaturation caused by freezing.

[0003] 2. Description of the Prior Art

[0004] In the field of genetic engineering, biochemistry, food andpharmaceutical industries, and the like, various proteins includingenzymes are commonly preserved by freezing.

[0005] Generally, proteins are known to change their high-dimensionalstructure upon freezing and thawing. When such denaturation of proteinsassociated with the freezing (occasionally called freeze denaturationhereinafter) occurs, protein activity may be decreased or completelylost, which causes problems in storing proteins.

[0006] Conventionally, in order to prevent such freeze denaturation ofproteins, bovine serum albumin (BSA), glycerol, sugars, or the like areusually added to the proteins to be kept frozen for storage.

[0007] However, when the abovementioned additives are used, bovine serumalbumin (BSA) is limited in its supply and generally very expensive.Further, serums derived from animals cannot sufficiently ensure thesafety because of their possible risk of viral infection. Furthermore,glycerol, sugars or the like must be added at a concentration as high as20% or so, which affects activity of proteins of interest. Further inthis case, a process to remove the abovementioned additives may berequired after freezing and thawing depending on the kind of proteins oruse thereof, which may require a complicated removing step or mayincrease the cost. Also for this reason, use of these additives has notbeen desirable.

[0008] Accordingly, there is still a need for means to preventfreeze-induced decrease in protein activity or inactivation of proteinsduring freezing/thawing cycles.

[SUMMARY OF THE INVENTION]

[0009] The present inventors have recently found that polypeptideshaving a specific amino acid repeat sequence are effective in theprevention of decrease in activity or inactivation of proteins, such asenzymes, associated with freeze denaturation. The present invention isbased on this finding.

[0010] Accordingly, an objective of the present invention is to providea means to prevent freeze-induced decrease in protein activity.

[0011] Further, a method according to the present invention is a processfor preventing freeze-induced decrease in protein activity comprisingadding a polypeptide comprising the following amino acid sequence (I) toa protein to be frozen: (SEQ ID NO: 1)Ser-Ser-Thr-Gly-Ser-X1-Ser-X2-Thr-Asp-X3-X4-X5-X6-X7-X8-Gly-Ser-X9-Thr-Ser-Gly-Gly-Ser-Ser-Thr-Tyr-Gly-Tyr-Ser-Ser-X10-X11-X12-X13-Gly-X14-Val (I)

[0012] wherein,

[0013] X1 represents Ser or Thr,

[0014] X2 represents Asn or Thr,

[0015] X3 represents Ser or Ala,

[0016] X4 represents Asn or Ser,

[0017] X5 represents Ser or Thr,

[0018] X6 represents Asn, Asp, or Lys,

[0019] X7 represents Ser, Asn, or Lys,

[0020] X8 represents Ala, Thr, or Val,

[0021] X9 represents Ser, or Arg,

[0022] X10 represents Asn, Ser, Asp, or Arg,

[0023] X11 represents Ser, Asn, His, or Cys,

[0024] X12 represents Arg or Gly,

[0025] X13 represents Asp or Gly, and

[0026] X14 represents Ser or Arg.

[0027] Further, the present invention provides use of a polypeptidecomprising the abovementioned amino acid sequence (I) to produce anagent for preventing freeze-induced decrease in protein activity.

[0028] According to the method of the present invention, decrease inprotein activity during freezing/thawing of proteins such as enzymes canbe prevented, stability of proteins during storage can be improved, andthe range of application of the proteins after storage by freezing canbe extended. The method according to the present invention is extremelyuseful in the field of food industry, pharmaceutical industry, and thelike.

[BRIEF DESCRIPTION OF THE DRAWINGS]

[0029]FIG. 1 shows photographs demonstrating the result of Coomassiestaining and the result of detection of histidine tags by Ni-NTA, whichconfirms the production of SP by gene expression in Escherichia coli.

[0030]FIG. 2 shows a photograph of the result for confirming the purityof SP produced by expression in Escherichia coli.

[0031]FIG. 3 shows the result of a test to evaluate activity of SP inprotecting enzyme against freezing stress.

[DETAILED DESCRIPTION OF THE INVENTION]

[0032] A process for preventing freeze-induced decrease in proteinactivity according to the present invention comprises adding apolypeptide comprising the abovementioned amino acid sequence (I) to aprotein to be frozen. In the abovementioned amino acid sequence (I), X1to X14 independently represent as follows:

[0033] X1 represents Ser or Thr, preferably Ser,

[0034] X2 represents Asn or Thr, preferably Asn,

[0035] X3 represents Ser or Ala, preferably Ala,

[0036] X4 represents Asn or Ser, preferably Asn,

[0037] X5 represents Ser or Thr, preferably Ser,

[0038] X6 represents Asn, Asp, or Lys, preferably Asn,

[0039] X7 represents Ser, Asn, or Lys, preferably Asn

[0040] X8 represents Ala, Thr, or Val, preferably Ala

[0041] X9 represents Ser, or Arg, preferably Ser,

[0042] X10 represents Asn, Ser, Asp, or Arg, preferably Asn,

[0043] X11 represents Ser, Asn, His, or Cys, preferably Ser,

[0044] X12 represents Arg or Gly, preferably Arg,

[0045] X13 represents Asp or Gly, preferably Asp, and

[0046] X14 represents Ser or Arg, preferably Ser.

[0047] In the present invention, said polypeptide can further comprise ahomologue of the abovementioned amino acid sequence (I). The term“homologue” herein used means a polypeptide having an amino acidsequence of the abovementioned amino acid sequence (I) in which one ormore (preferably one or several) amino acids are deleted, substituted,inserted, or added, still having a function for preventingfreeze-induced decrease (or inactivation) in protein activity(occasionally referred to as cryoprotective activity in thisspecification).

[0048] In a preferred embodiment of the present invention, theabovementioned amino acid sequence (I) is preferably the following aminoacid sequence (II), namely said polypeptide according to the presentinvention preferably comprises the following amino acid sequence (II):

[0049] Ser-Ser-Thr-Gly-Ser-Ser-Ser-Asn-Thr-Asp-Ser-Asn-Ser-Asn-Ser-

[0050] Ala-Gly-Ser-Ser-Thr-Ser-Gly-Gly-Ser-Ser-Thr-Tyr-Gly-Tyr-Ser-

[0051] Ser-Asn-Ser-Arg-Asp-Gly-Ser-Val (II) (SEQ ID NO: 2).

[0052] Said polypeptide according to the present invention can typicallyprevent protein denaturation, typically decrease in protein activitycaused by freezing that may occur upon freezing of a protein ofinterest, when added to said protein of interest.

[0053] The term “preventing freeze-induced decrease in protein activity”herein means preventing decrease in protein activity or inactivation ofproteins that may be caused when a protein of interest is frozen orfrozen and thawed. The term also implies maintaining activity of saidprotein, stabilizing said protein, and preventing protein denaturation.

[0054] In the present invention, “freezing” proteins typically meansthat proteins are maintained under usual freezing storage conditions,for example, in refrigeration below 0° C.

[0055] In the present invention, proteins of to be frozen are notparticularly restricted and can be any proteins as long as theiractivity is decreased or lost by freezing. In the present invention,preferable examples of such proteins are enzymes. Examples of suchenzymes include those important in industry, such as proteases,amylases, cellulases, lipases, restriction enzymes, and modificationenzymes.

[0056] Furthermore, the present invention also provides use of apolypeptide comprising the abovementioned amino acid sequence (I) forthe production of agents for preventing freeze-induced decrease inactivity of proteins.

[0057] Here, “agents for preventing decrease in activity” are thosewhich can prevent decrease in activity of proteins or inactivation ofproteins that may be caused upon freezing or thawing of the proteins ofinterest. Forms, doses or the like of the agents are not particularlyrestricted.

[0058] Further, the polypeptide comprising the abovementioned amino acidsequence (I) can, if present in a cell, control the stress associatedwith dehydration of the cell which affects the cell. Namely, saidpolypeptides can protect the cell from the dehydration stress(occasionally referred to as “protective function against dehydrationstress” in this specification). In other words, the polypeptideaccording to the present invention may further be able to renderdehydration stress tolerance to the cell. Examples of such cell (ortarget cell) include cells of microorganisms such as Escherichia coli,Bacillus subtilis and yeast, fungi, insects, animals, and plants.

[0059] Thus, according to another embodiment of the present invention,there are provided a process for rendering dehydration stress toleranceto a target cell comprising introducing a polypeptide comprising theamino acid sequence (I) to the target cell for the accumulation therein.Preferably, this process for rendering dehydration stress tolerance tothe target cell comprises transforming the target cell using arecombinant vector (to be explained later) comprising a DNA encoding apolypeptide comprising the amino acid sequence (I). Further, in thisspecification, the terms “DNA” and “gene” may occasionally be used formeaning the same.

[0060] According to a preferred embodiment of the present invention,said polypeptide preferably comprises a repetitive sequence in which theabovementioned amino acid sequence (I) is repeated at least twice. Useof the polypeptide having such a repetitive sequence can further improvethe effectiveness for preventing decrease in protein activity by themethod according to the present invention.

[0061] Accordingly, the number of repeat of the amino acid sequence (I)in a polypeptide of the present invention is preferably at least 2. Thegreater number of repeat is preferable to attain the more remarkableabovementioned effectiveness. However, the number of the repeat ispreferably 2 to 8, more preferably 2 to 6, and most preferably 2 to 4,taking possible cost increase due to the complicated repeating processand the compatibility upon the introduction into a cell intoconsideration.

[0062] A polypeptide comprising a repetitive sequence in which theabovementioned amino acid sequence (I) is repeated at least twice can beobtained, for example, as follows:

[0063] First, a DNA encoding a repetitive sequence in which the aminoacid sequence (I) is repeated twice is designed and then synthesizedusing an ordinary DNA synthesizer. Here, it is desirable to placerestriction enzyme recognition sites on both terminals of theabovementioned sequence to link to a vector and a translation stop codonat the 3′ terminal. Further, if it is not desirable to obtain as anentire length of DNA chain considering reliability and operability of aDNA synthesizer or a DNA purification method, segmented fragments may befirst prepared and then linked together to obtain the entire length ofDNA. In this way, a DNA encoding a repetitive sequence of interest canbe obtained. Next, the DNA thus obtained is expressed to obtain apolypeptide, for example, using a method described hereinafter. Thus, apolypeptide comprising a repetitive sequence (for example, the followingsequence (III)), in which the amino acid sequence (I) is repeated twice,can be obtained.

[0064] Amino acid sequence (III): (SEQ ID NO: 3)Ser-Ser-Thr-Gly-Ser-Ser-Ser-Asn-Thr-Asp-Ser-Asn-Ser-Asn-Ser-Ala-Gly-Ser-Ser-Thr-Ser-Gly-Gly-Ser-Ser-Thr-Tyr-Gly-Tyr-Ser-Ser-Asn-Ser-Arg-Asp-Gly-Ser-Val-Ser-Ser-Thr-Gly-Ser-Ser-Ser-Asn-Thr-Asp-Ser-Asn-Ser-Asn-Ser-Ala-Gly-Ser-Ser-Thr-Ser-Gly-Gly-Ser-Ser-Thr-Tyr-Gly-Tyr-Ser-Ser-Asn-Ser-Arg- Asp-Gly-Ser-Val.

[0065] The sequence consisting of 38 amino acids of the abovementionedamino acid sequence (I) is hereinafter referred to as “SP” and thepolypeptide consisting of two repeats thereof is occasionally calledSP2.

[0066] Further in the present invention, a DNA encoding SP4 having 4repetitive SPs can be obtained by obtaining a DNA in which specificrestriction enzyme sites are added to both terminals of a DNA encodingSP2 synthesized by the PCR method using specific primers and thenlinking these restriction enzyme sites using restriction enzymes. Thus,by using the same technique, a DNA encoding SP6, a DNA encoding SP8, andthe like can be obtained one by one, and DNAs encoding more repetitivesequences can be obtained in necessary. As described above, DNAs thusobtained can be expressed to obtain polypeptides such as SP4, SP6, andSP8.

[0067] A polypeptides comprising a repetitive sequence having at leasttwo repeats of the amino acid sequence (I) can be obtained in such amanner as described above; however, alternatively, the abovementionedpolypeptide of interest can be obtained by first obtaining more than onepolypeptides having the amino acid sequence (I) using, for example, themethod described hereinafter and then linking them one another using aconventional chemical method.

[0068] In the present invention, a polypeptide comprising theabovementioned amino acid sequence (I) can either be produced usingvarious conventional synthesis methods or be derived from nature. As theamino acid sequence of this polypeptide has been determined, it can beobtained by synthesizing its whole sequence, or partially using asequence derived from nature and further synthesizing based on thesequence.

[0069] According to a preferred embodiment of the present invention,said polypeptide can be derived from a natural protein, sericin, andaccordingly be obtained from this sericin using conventional geneticengineering. Natural sericin can be obtained, for example, from silkgland tissue of silkworm, cocoons, or raw silk. In the presentinvention, sericin implies hydrolysis products of this protein inaddition to sericin itself.

[0070] Further, in the present invention, in cases where a DNA encodinga polypeptide comprising the abovementioned amino acid sequence (I) isavailable or can be produced, the polypeptide can be produced in atransformed cell obtained by transforming a host cell with this DNA.More specifically, a polypeptide according to the present invention canbe produced by culturing a transformant obtained by transforming a hostcell using a DNA, in particular a recombinant vector, which contains aDNA fragment encoding said polypeptide according to the presentinvention in a form amplifiable and replicable in the host cell. Namely,in the present invention, what is called a host-vector system can beused for producing said polypeptide. In the present invention, uponapplying such a host-vector system, various methods commonly used inthis field for the construction of expression vectors (recombinantvectors) and the transformation can be used.

[0071] Thus, according to another preferred embodiment of the presentinvention, the abovementioned polypeptide is obtained by a methodcomprising:

[0072] preparing a recombinant vector comprising a DNA encoding apolypeptide comprising the amino acid sequence (I),

[0073] obtaining a transformed cell by transforming a cell using saidrecombinant vector,

[0074] culturing said transformed cell, and

[0075] recovering a polypeptide from the resulting cells and/or culturethereof.

[0076] In the present invention, a DNA containing a gene encoding theabovementioned polypeptide, particularly a recombinant vector, can beobtained by incorporating a DNA fragment encoding the abovementionedpolypeptide into a commonly used vector system. In the presentinvention, it is preferable that this vector contains the encoding DNAfragment in a repetitive form as mentioned above.

[0077] A vector to be used in the present invention can be selected fromcommonly used vectors in which a host-vector system is established, suchas plasmids, viruses, phages, and cosmid vectors, depending on the kindof a host cell to be used. More specifically, for example, a pBR-, pUC-or pQE-based plasmid, or λ-phage bacteriophage is used when Escherichiacoli is a host cell, a pUB-based plasmid is used when Bacillus subtilisis a host cell, and a YEp- or YCp-based vector is used when yeast is ahost cell. A vector to be used in the present invention is preferably aplasmid.

[0078] A plasmid usable in the present invention preferably contains amarker for selecting a transformant. Examples of such marker includegenes conferring resistance to drugs, such as ampicillin and kanamycin,and genes complementing a nutritional requirement. Further, in thepresent invention, restoration of β-galactosidase activity by a specificpeptide generated by a vector DNA such as a plasmid and a peptideencoded in a host cell can also be used as a selection marker.

[0079] Further in the present invention, a DNA to be used as saidrecombinant vector preferably has DNAs necessary for expressing apolypeptide comprising the abovementioned amino acid sequence (I), suchas a promoter, transcription start signal, translation stop signal,transcription control signal such as transcription termination signal,and translation control signal.

[0080] The present invention provides a transformant obtained bytransforming a host cell with the abovementioned recombinant vector.

[0081] In the present invention, any host cell can be used as long asits host-vector system is established. Examples of such host cellinclude Escherichia coli, Bacillus subtilis, yeasts and fungi.

[0082] When a host cell is Escherichia coli, Bacillus subtilis, yeast orfungus, a secretion vector that extracellularly secretes theabovementioned polypeptide of interest can be used as a vector.

[0083] According to still another embodiment of the present invention,the abovementioned polypeptide can be a chimeric protein in which apolypeptide comprising the abovementioned amino acid sequence (I) and aheterologous polypeptide (for example, another functional protein) arehybridized.

[0084] A chimeric protein can be produced by expressing a DNA encodingthe chimeric protein made by DNA fusion using a DNA encoding apolypeptide comprising the abovementioned amino acid sequence (I) and aDNA encoding a heterologous polypeptide.

[EXAMPLE]

[0085] The present invention is further illustrated by the followingexamples that are not intended as a limitation of the invention.

Chemical Synthesis of Gene Fragments Encoding a Polypeptide havingCryoprotective Activity

[0086] A gene (hereinafter called serD) encoding a polypeptideconsisting of the abovementioned amino acid sequence of SP2 (theabovementioned sequence (III) (SEQ ID NO: 3)) was designed. Here, arecognition site (Ile-Glu-Gly-Arg) for protease (Factor Xa) was locatedat the N-terminal of SP2 to cleave another fused polypeptide.Furthermore, the abovementioned gene was designed, taking the codonusage frequency of Escherichia coli (Ikemura, T. and Ozeki, H., ColdSpring Harbor Symp. Quant. Biol., 47, 1087(1983)) into consideration, tolocate restriction enzyme recognition sites (PstI, EcoRI) for thelinkage to a vector at both terminals (5′ and 3′ terminals) of serD andto add two translation stop codons at the 3′ terminal side.

[0087] Next, the designed gene was chemically synthesized usingphosphoramidite chemistry on a DNA synthesizer (Applied Biosystems). Inthis case, taking reliability and operability of current DNAsynthesizers and DNA purification methods into consideration, the DNAwas synthesized as divided fragments each having about 60 to 70 bases.

[0088] Said repetitive unit SP is consisted of 38 amino acid residues,namely 114 bases. Taking stability of gene products, a DNA encoding SP2(serD) in which a repetitive unit of 38 amino acid residues is repeatedtwice is used as a base unit of synthesized DNA. Therefore, a gene of atleast 228 bases is necessary. Furthermore, since the DNA has to beincorporated as a double-stranded chain, twice the amount of DNA has tobe synthesized.

[0089] In practice, the introduction of stop codons and the restrictionenzyme sites for linkage of each fragment to a plasmid are alsonecessary for the synthetic gene encoding SP2. Therefore the entirety ofgene encoding SP2 was divided into 4 fragments, so that 4 front-and-backpairs, i.e., the total of 8, of DNA chains were synthesized.

[0090] The 8 DNA fragments synthesized are as follows: serD fragment (1)5′-GTGATCAATCGAAGGTCGCTCGAGTACTGGTTCTTCTTCTAACACCGACTCTAACT (SEQ ID NO:4) CTAAC-3′ serD fragment (2)5′-TCTGCTGGTTCTTCTACCTCTGGTGGTTCTTCTACCTACGGTTACTCTTCTAACT (SEQ ID NO:5) CTCGTGACGGTTCT-3′ serD fragment (3)5′-GTTTCTTCTACCGGTTCTTCTTCTAACACCGACTCTAACTCTAACTCTGCTGGTT (SEQ ID NO:6) CTTCTACCTC-3′ serD fragment (4)5′-TGGTGGTTCTTCTACCTACGGTTACTCTTCTAACTCTCGTGACGGATCCGTTTAA (SEQ ID NO:7) TAGCTGAGCG-3′ serD fragment (1′)5′-CAGAGTTAGAGTTAGAGTCGGTGTTAGAAGAAGAACCAGTACTCGAGCGACCTTC (SEQ ID NO:8) GATTGATCACTGCA-3′ serD fragment (2′)5′-AAACAGAACCGTCACGAGAGTTAGAAGAGTAACCGTAGGTAGAAGAACCACCAGA (SEQ ID NO:9) GGTAGAAGAACCAG-3′ serD fragment (3′)5′-ACCAGAGGTAGAAGAACCAGCAGAGTTAGAGTTAGAGTCGGTGTTAGAAGAAGAA (SEQ ID NO:10) CCGGTAGAAG-3′ serD fragment (4′)5′-AATTCGCTCAGCTATTAAACGGATCCGTCACGAGAGTTAGAAGAGTAACCGTAGG (SEQ ID NO:11) TAGAAGAACC-3′

Construction of Gene (serD) Encoding SP2

[0091] The eight fragments (about 70 bp) synthesized as mentioned abovewere made into double-stranded chains by an annealing process withfragments having each other a complementary sequence portion to obtainfour double-stranded DNA fragments constructing serD.

[0092] Further, the 5′ terminal of the synthesized gene fragment wasphosphorylated using T4 polynucleotide kinase, since no phosphoric waspresent at the 5′ terminal of the DNA encoding the oligonucleotideobtained by the chemical synthesis.

[0093] Namely, the following components were placed into a tube(manufactured by Eppendolf Co.), reacted at 37° C. for 1 hour, and thenheated at 70° C. for 5 minutes to inactivate the enzyme. Individualoligonucleotide 100 pmole (in water) 7.5 μl 10 × buffer* 1 μl 10 mM ATP1 μl T4 polynucleotide kinase (Takara Shuzo Co., Ltd.) 10 μl 0.5 μl (5units)

[0094] [* 10×buffer: 650 nM Tris-HCl (pH 7.6), 100 mM MgCl₂, 150 mMdithiothreitol, 10 mM Spermidine.]

[0095] A 5 μl portion of each of the DNAs was mixed in a tube(manufactured by Eppendolf Co.) and reacted for linkage at 16° C. for 30minutes using a Takara ligation kit version II (Takara Shuzo Co., Ltd.)to adjacently link the four DNA fragments. After the reaction, agarosegel electrophoresis was carried out and a DNA fragment of about 270 bpwas recovered from the gel. The resulting DNA fragment had a PstIrecognition site at the 5′ terminal and an EcoRI recognition site at the3′ terminal.

[0096] Further, said DNA fragment was mixed with an appropriate amountof plasmid pUC19 (Yanisch-Perron, C. et al, Gene, 33,103(1985))previously cleaved with PstI and EcoRI, and ligation reactionwas carried out at 16° C. for 1 hour using a Takara ligation kit versionII (Takara Shuzo Co., Ltd.).

[0097] Next, the resulting reaction mixture was introduced intoEscherichia coli strain JM109 (recAl,Δlac-proAB, endAl, gryA96, thi-1,hsdR17, supE44, relAl, λ⁻, (F′traD36, proAB, lacI q Z ΔM15)).

[0098] Further, this E. coli strain JM109 is a strain in which upontransformation of pUC-based plasmid DNA or transduction of M13 phagevector DNA, a lacZα peptide generated from the vector DNA and lacZΔM15encoded by JM109F′ restore β-galactosidase activity, which facilitates aselection of recombinants.

[0099] Accordingly, in a medium containing IPTG(isopropyl-β-D-thiogalactopyranoside) and X-Gal(5-bromo-4-chloro-3-indole-β-D-galactoside), cells of this strain JM109carrying plasmid pUC19 form blue colonies showing β-galactosidaseactivity. On the other hand, since β-galactosidase activity cannot berestored in strain JM109 carrying a recombinant plasmid in which aforeign DNA fragment is inserted, cells of this strain form whitecolonies. The recombinant plasmids can thereby be selected.

[0100] Accordingly, plasmids were prepared from white colonies formedand subjected to DNA sequencing (Sanger, F. et al, J. Mol. Biol., 143,161 (1980)) to select a clone having a serD base sequence (fragment)exactly the same as designed.

[0101] The recombinant plasmid having the serD gene thus obtained isherein called pUC-serD.

Polymerization of SP

[0102] Polymerization of SP was carried out as follows.

[0103] pET-serD

[0104] A fragment containing serD obtained by cleaving theabovementioned pUC-serD with Bcl1 and Bpu1102I was mixed with vectorpET3a (Novagen) previously cleaved with restriction enzymes BamHl andBpu1102I, and ligation reaction was carried out at 16° C. for 1 hourusing a Takara ligation kit version II (Takara Shuzo Co., Ltd.).

[0105] Next, the resulting reaction mixture was introduced into E. colistrain JM109 as described above. A plasmid was prepared from atransformant thus obtained, subjected to DNA sequencing and thenconfirmed to have the sequence of serD.

[0106] The recombinant plasmid having the serD gene thus obtained isherein called pET-serD.

[0107] pET-serT

[0108] Next, a serD gene having XhoI sites added at both terminals ofthe synthesized serD gene for SP2 was obtained by the PCR method. ThePCR reaction was carried out by an ordinary method using an Ex Taq(Takara Shuzo Co., Ltd.). Primers used herein are as follows:5′-AAGGTCGCTCGAGTACCGGT-3′ (SEQ ID NO: 12) 5′-CGCTCAGACTCGAGACAGAT-3′(SEQ ID NO: 13)

[0109] Next, the serD gene having the added XhoI sites at both terminalswas linked to the XhoI site of pET-serD utilizing the restriction enzymesites to construct plasmid pET-serT having a gene encoding SP4consisting of 4 repetitive SPs.

[0110] pET-serH

[0111] Similarly, a serD gene in which a BamHI site was added to the 5′terminal was obtained by the PCR method.

[0112] By utilizing the restriction enzyme sites, the resulting serD waslinked to the BamHI site of pET-serT to construct plasmid pET-serHhaving a gene encoding SP6 consisting of 6 repetitive SPs.

[0113] Primers herein used for introducing the BamHI site into the serDgene are as follows: 5′-GTTTTCCCAGTCACGAC-3′ (SEQ ID NO: 14)5′-ATCGGATCCGTCTCGAGTACT-3′ (SEQ ID NO: 15)

[0114] pET-serO

[0115] Similarly, a serD gene in which a ScaI site was added to the 3′terminal was obtained by the PCR method.

[0116] By utilizing the restriction enzyme sites, the resulting serD waslinked to the ScaI site of pET-serH to construct plasmid pET-serO havinga gene encoding SP8 consisting of 8 repetitive SPs.

[0117] Primers herein used for introducing the ScaI site into the serDgene are as follows: 5′-CAGGAAACAGATATGAC-3′ (SEQ ID NO: 16)5′-GCTAGTACTCGAAACGGATC-3′ (SEQ ID NO: 17)

Construction of Expression Plasmid for E. coli

[0118] Construction of pQE-NHserD

[0119] A fragment containing serD obtained by cleaving pUC-serD withPstI and EcoRI was mixed with plasmid pBSIISK+ previously cleaved withPstI and EcoRI, and ligation reaction was carried out at 16° C. for 1hour using a Takara ligation kit version II (Takara Shuzo Co., Ltd.).

[0120] Next, the resulting reaction mixture was introduced into E. colistrain JM109 as described above. A plasmid was prepared from atransformant thus obtained, from which it was confirmed that the serDgene (about 270 bp) was properly inserted.

[0121] The recombinant plasmid having the serd gene thus obtained isherein called pBS-serD.

[0122] Next, a fragment containing serD obtained by cleaving pBS-serDwith BclI and HindIII was mixed with a high-level expression vectorpQE30 for E. coli (Qiagen) previously cleaved with BclI and HindIII, andligation reaction was carried out at 16° C. for 1 hour using a Takaraligation kit version II (Takara Shuzo Co., Ltd.).

[0123] Next, the resulting reaction mixture was introduced into E. colistrain JM109 as described above.

[0124] A plasmid was prepared from a transformant thus obtained, fromwhich it was confirmed that the serD gene (about 270 bp) was properlyinserted.

[0125] The recombinant plasmid having the serD gene thus obtained isherein called pQE-NHserD.

[0126] Here, a 6×His tag was designed to be located at the N-terminal ofSP2. The amino acid sequence of the expressed polypeptide is shown inSEQ ID NO: 18.

Construction of pQE-NHLserT, pQE-NHLserH, and pQE-NHLserO

[0127] The abovementioned plasmids pET-serT, pET-serH, and pET-serO werecleaved with NheI to obtain fragments containing serT, serH, and serO,and these fragments were each linked to the XbaI site of PBSIISK+ toconstruct recombinant plasmids. These recombinant plasmids were thencleaved with ScaI and HindIII to prepare fragments containing genesserT, serH, and serO encoding SP4, SP6, and SP8, respectively. The genefragments thus prepared were each linked to a high-level expressionvector pQE30 for E. coli to construct individual expression plasmidspQE-NHLserT, pQE-NHLserH and pQE-NHLserO.

[0128] Here, 6×His tags were designed to be located at the N-terminalsof SP4, SP6, and SP8.

[0129] The amino acid sequences of the expressed polypeptides are shownin SEQ ID NO: 19, SEQ ID NO: 20, and SEQ ID NO: 21.

Induction and Confirmation of Expression of Gene Encoding SP in E. coli

[0130] Preparation of E. coli Transformant

[0131] Expression vectors pQE-NHserD, pQE-NHLserT, pQE-NHLserH, andpQE-NHLserO, into which genes encoding SPs were incorporated, and thesole vector pQE as a control were each introduced into E. coli strainJM109 (recAl,Δlac-proAB, endal, gryA96, thi-1, hsdR17, supE44, relAl,λ⁻, (F′traD36, proAB, lacI q Z ΔM15)). Since the expression vector pQEfor E. coli carries an ampicillin resistance gene as a selection marker,transformants were selected as ampicillin resistant colonies.

[0132] Induction of Expression

[0133]E. coli JM109 transformants each carrying an expression plasmidinto which a gene encoding SP was incorporated were cultured in a M9+2%casamino acid medium supplemented with 50 μg/ml ampicillin at 37° C.overnight with shaking. The resulting culture was inoculated into thesame medium at a concentration of 2%, and cultivation was continued at37° C. with shaking.

[0134] IPTG (isopropyl β-D-thiogalactopyranoside) was added to theculture thus obtained at a final concentration of 1 mM when theabsorbance at 610 nm reached 0.3 to 0.5, and the cultivation wascontinued for 4 hours.

[0135] Confirmation of Expression

[0136] After cultivation, the culture supernatant was removed bycentrifugation, and the resulting cells were resuspended in a buffersolution [50 mM Na-phosphate(pH7.8), 300 mM NaCl] of 1/10 the volume ofthe culture medium. Then, the cells were ruptured by sonication (200W,about 30 minutes) to prepare a cell-free extract.

[0137] A portion of the cell-free extract was subjected toSDS-polyacrylamide gel electrophoresis by an ordinary method. Coomassiestaining of the resulting gel confirmed the expressed SP as a thickband.

[0138] Further, the SDS-polyacrylamide gel electrophoresis pattern wastransferred to a nitrocellulose membrane, after which the histidinehexamer (6×His) tag added to the N-terminal side of SP was detected bychemical color reaction using HRP-labeled Ni-NTA (nitrilotriacetic acid)(Qiagen), and thus the production of targeted SP was confirmed.

[0139] Further, the result of N-terminal amino acid sequencing of thepeptide using Edman degradation confirmed that it had the amino acidsequence as designed.

[0140] The results of the abovementioned Coomassie staining andhistidine tag detection by Ni-NTA are shown in FIG. 1.

[0141] Purification of SP

[0142] Since peptides produced by expression are highly hydrophilic andvirtually do not form high-dimensional structure, they are notprecipitated by treating at 100° C. for 10 minutes. By utilizing thisproperty, the cell-free extract prepared by sonication was treated at100° C. for 10 minutes and then centrifuged at 6,500 rpm for 5 minutesto precipitate denatured proteins derived from E. coli and recover SPfrom the supernatant as a soluble fraction.

[0143] Next, SP was purified from the supernatant of the cell-freeextract using QIAexpress Ni-NTA Protein Purification System (Qiagen).This QIAexpress Ni-NTA Protein Purification System can purify a proteinhaving a histidine hexamer (6×His) tag utilizing its high affinity withNi-NTA (nitrilotriacetic acid).

Test for Evaluating Cryoprotective Activity on Enzyme

[0144] Preparation of SP

[0145] An E. coli JM109 transformant carrying an expression plasmid(pQE-NHserD) into which a gene encoding SP was incorporated was culturedin a M9+2% casamino acid liquid medium supplemented with 50 μg/mlampicillin at 37° C. overnight with shaking. Next, the resulting culturewas inoculated into the same medium at a concentration of 2%, andcultivation was continued at 37° C. with shaking.

[0146] IPTG (isopropyl β-D-thiogalactopyranoside) was added at a finalconcentration of 1 mM when the absorbance at 610 nm reached 0.3 to 0.5to induce gene expression, and the cultivation was continued for 4hours.

[0147] After cultivation, the culture supernatant was removed bycentrifugation, and the resulting cells were resuspended in a buffersolution [50 mM Na-phosphate (pH7.8), 300 mM NaCl] of 1/10 the volume ofthe culture medium. Then, the cells were ruptured by sonication (200W,about 30 minutes) to prepare a cell-free extract.

[0148] Using a portion of the cell-free extract, SDS-polyacrylamide gelelectrophoresis and Coomassie staining was carried out according toordinary methods to confirm SP expression.

[0149] Utilizing the abovementioned property of SP produced byexpression, the cell-free extract prepared by sonication was treated byheating at 100° C. for 10 minutes and then centrifuged at 6,500 to12,000 rpm for 5 to 20 minutes to precipitate denatured proteins derivedfrom E. coli and recover SP from the supernatant as a soluble fraction.

[0150] Next, SP contained in the supernatant after heat treatment wasfurther purified using QIAexpress Ni-NTA Protein Purification System(Qiagen). In this QIAexpress Ni-NTA Protein Purification System, aprotein having a histidine hexamer (6×His) tag is absorbed utilizinghigh affinity with Ni-NTA (nitrilotriacetic acid) and then eluted with a0.02-1.0 M imidazole solution.

[0151] The purified SP was confirmed by SDS-polyacrylamide gelelectrophoresis and Coomassie staining (FIG. 2). Further, the SPconcentration was measured by the BCA method extensively used forprotein quantification.

Evaluation Test (Cryoprotective Activity on LDH)

[0152] SP produced by the expression in E. coli was measured forprotective activity against loss of enzymatic activity caused byfreezing/thawing.

[0153] Lactate dehydrogenase (LDH) was used as a model enzyme. Lactatedehydrogenase (LDH) is an enzyme that acts in a process of producingL-lactic acid from pyruvic acid in a glycolytic pathway. LDH is known tobe highly sensitive to freezing stress and tend to lose its activity byfreezing/thawing processes (K. Goller, E. A. Galinski, Journal ofMolecular Catalysis B: Enzymatic 7 (1999) pp37-45).

[0154] LDH reaction system is shown as follows:

L-lactic acid+AND⁺⇄Pyruvic acid+NADH+H⁺

[0155] A commercial LDH (5,000 U/ml) (Oriental Yeast Co., Ltd., frompigheart) was dilutedwith a 50 mMpotassiumphosphate buffer solution toprepare an about 250-unit enzyme solution. The enzyme solution thusprepared was dialyzed at 4° C. overnight to completely remove ammoniumsulfate and the like contained in the commercial enzyme solution.

[0156] The dialyzed enzyme solution was diluted with a 50 mM potassiumphosphate buffer solution to prepare an about 4-unit LDH solution.

[0157] A potassium phosphate buffer solution and the LDH solution weremixed and the resulting mixture was preincubated at 25° C., after whichsodium pyruvate and NADH were quickly added and change in absorbance at340 nm was measured for 5 minutes using a spectrophotometer (Beckman,DU640).

[0158] Composition of a reaction solution for LDH activity measurementand measuring conditions for the spectrophotometer are shown below.[Composition of reaction solution] 0.1 M Potassium phosphate buffer (pH7.0) 3.00 ml 25.4 mM Sodium pyruvate 0.10 ml NADH [10 mg/ml (10 mMTris)] 0.05 ml Lactate dehydrogenase (LDH from pig heart, 4 U/ml) 0.02ml 3.17 ml

[0159] [Measuring Conditions]

[0160] Wavelength: 340 nm

[0161] Optical path length: 1 cm

[0162] Temperature: 25° C.

[0163] LDH activity was determined from change in NADH (change inabsorbance at 340 nm)using the following formula (i)

(ΔA/min·V·D)/(ε·d·v)=IU/ml  (i)

[0164] wherein

[0165] ΔA/min=change in absorbance at 340 nm per minute,

[0166] V=final liquid volume (3.17 ml),

[0167] D=final dilution rate,

[0168] e=molecular absorption coefficient of NADH at 340 nm (6.3×10³l·mole⁻¹·cm⁻¹),

[0169] d=optical path length (1 cm), and

[0170] v=volume of enzyme solution (0.02 ml).

[0171] The purified SP was added at concentrations of 0.01% and 0.05% tothe LDH solutions (about 4 unit/ml) prepared by the abovementionedmethod.

[0172] To prepare control LDH solutions for comparison, bovine serumalbumin (BSA) (Sigma, Albumin bovine fraction V) that is used as anagent for preventing freeze-induced decrease in protein activity wasadded at a concentration of 0% (potassium phosphate buffer only), 0.01%,and 0.1% to the LDH solutions.

[0173] The sample LDH solutions thus prepared are summarized as follows:

[0174] LDH [4 unit/ml],

[0175] LDH [4 unit/ml]+0.01% SP

[0176] LDH [4 unit/ml]+0.05% SP

[0177] LDH [4 unit/ml]+0.01% BSA

[0178] LDH [4 unit/ml]+0.05% BSA

[0179] Next, a 100 μl portion of each of the prepared samples wasdispensed into a 1.5-ml test tube and frozen for 1 minute with liquidnitrogen. Then, after thawing at 30° C. for 5 minutes, LDH activity wasmeasured by the abovementioned method. Remaining LDH activity for eachsample after repeating freezing/thawing cycles was expressed by settingthe LDH activity before freezing to 100%.

[0180] The remaining LDH activity in the samples without SP was markedlydecreased by repetitive freezing/thawing cycles and was about 3% after 8freezing/thawing cycles, while the sample with SP had about 90%remaining activity.

[0181] The abovementioned results are shown in FIG. 3.

1 21 1 38 PRT Artificial CONSENSUS SEQUENCE 1 Ser Ser Thr Gly Ser XaaSer Xaa Thr Asp Xaa Xaa Xaa Xaa Xaa Xaa 1 5 10 15 Gly Ser Xaa Thr SerGly Gly Ser Ser Thr Tyr Gly Tyr Ser Ser Xaa 20 25 30 Xaa Xaa Xaa Gly XaaVal 35 2 38 PRT Artificial FRAGMENT FOR RESISTANCE AGAINST DEHYDRATIONSTRESS 2 Ser Ser Thr Gly Ser Ser Ser Asn Thr Asp Ser Asn Ser Asn Ser Ala1 5 10 15 Gly Ser Ser Thr Ser Gly Gly Ser Ser Thr Tyr Gly Tyr Ser SerAsn 20 25 30 Ser Arg Asp Gly Ser Val 35 3 76 PRT Artificial FRAGMENT FORRESISTANCE AGAINST DEHYDRATION STRESS 3 Ser Ser Thr Gly Ser Ser Ser AsnThr Asp Ser Asn Ser Asn Ser Ala 1 5 10 15 Gly Ser Ser Thr Ser Gly GlySer Ser Thr Tyr Gly Tyr Ser Ser Asn 20 25 30 Ser Arg Asp Gly Ser Val SerSer Thr Gly Ser Ser Ser Asn Thr Asp 35 40 45 Ser Asn Ser Asn Ser Ala GlySer Ser Thr Ser Gly Gly Ser Ser Thr 50 55 60 Tyr Gly Tyr Ser Ser Asn SerArg Asp Gly Ser Val 65 70 75 4 61 DNA Artificial SYNTHETIC DNA 4gtgatcaatc gaaggtcgct cgagtactgg ttcttcttct aacaccgact ctaactctaa 60 c61 5 69 DNA Artificial SYNTHETIC DNA 5 tctgctggtt cttctacctc tggtggttcttctacctacg gttactcttc taactctcgt 60 gacggttct 69 6 65 DNA ArtificialSYNTHETIC DNA 6 gtttcttcta ccggttcttc ttctaacacc gactctaact ctaactctgctggttcttct 60 acctc 65 7 65 DNA Artificial SYNTHETIC DNA 7 tggtggttcttctacctacg gttactcttc taactctcgt gacggatccg tttaatagct 60 gagcg 65 8 69DNA Artificial SYNTHETIC DNA 8 cagagttaga gttagagtcg gtgttagaagaagaaccagt actcgagcga ccttcgattg 60 atcactgca 69 9 69 DNA ArtificialSYNTHETIC DNA 9 aaacagaacc gtcacgagag ttagaagagt aaccgtaggt agaagaaccaccagaggtag 60 aagaaccag 69 10 65 DNA Artificial SYNTHETIC DNA 10accagaggta gaagaaccag cagagttaga gttagagtcg gtgttagaag aagaaccggt 60agaag 65 11 65 DNA Artificial SYNTHETIC DNA 11 aattcgctca gctattaaacggatccgtca cgagagttag aagagtaacc gtaggtagaa 60 gaacc 65 12 20 DNAArtificial serD GENE FOR PCR PRIMER 12 aaggtcgctc gagtaccggt 20 13 20DNA Artificial serD GENE FOR PCR PRIMER 13 cgctcagact cgagacagat 20 1417 DNA Artificial serD GENE FOR PCR PRIMER 14 gttttcccag tcacgac 17 1521 DNA Artificial serD GENE FOR PCR PRIMER 15 atcggatccg tctcgagtac t 2116 17 DNA Artificial serD GENE FOR PCR PRIMER 16 caggaaacag atatgac 1717 20 DNA Artificial serD GENE FOR PCR PRIMER 17 gctagtactc gaaacggatc20 18 91 PRT Artificial FRAGMENT FOR RESISTANCE AGAINST DEHYDRATIONSTRESS 18 Met Arg Gly Ser His His His His His His Gly Ser Ile Glu GlyArg 1 5 10 15 Ser Ser Thr Gly Ser Ser Ser Asn Thr Asp Ser Asn Ser AsnSer Ala 20 25 30 Gly Ser Ser Thr Ser Gly Gly Ser Ser Thr Tyr Gly Tyr SerSer Asn 35 40 45 Ser Arg Asp Gly Ser Val Ser Ser Thr Gly Ser Ser Ser AsnThr Asp 50 55 60 Ser Asn Ser Asn Ser Ala Gly Ser Ser Thr Ser Gly Gly SerSer Thr 65 70 75 80 Tyr Gly Tyr Ser Ser Asn Ser Arg Gly Ser Val 85 90 19191 PRT Artificial FRAGMENT FOR RESISTANCE AGAINST DEHYDRATION STRESS 19Met Arg Gly Ser His His His His His His Gly Ser Ala Cys Glu Leu 1 5 1015 His Arg Gly Gly Gly Ala Ser Ser Met Thr Gly Gly Gln Gln Met Gly 20 2530 Arg Gly Ser Ile Glu Gly Arg Ser Ser Thr Gly Ser Ser Ser Asn Thr 35 4045 Asp Ser Asn Ser Asn Ser Ala Gly Ser Ser Thr Ser Gly Gly Ser Ser 50 5560 Thr Tyr Gly Tyr Ser Ser Asn Ser Arg Asp Gly Ser Val Ser Ser Thr 65 7075 80 Gly Ser Ser Ser Asn Thr Asp Ser Asn Ser Asn Ser Ala Gly Ser Ser 8590 95 Thr Ser Gly Gly Ser Ser Thr Tyr Gly Tyr Ser Ser Asn Ser Arg Asp100 105 110 Gly Ser Val Ser Ser Thr Gly Ser Ser Ser Asn Thr Asp Ser AsnSer 115 120 125 Asn Ser Ala Gly Ser Ser Thr Ser Gly Gly Ser Ser Thr TyrGly Tyr 130 135 140 Ser Ser Asn Ser Arg Asp Gly Ser Val Ser Ser Thr GlySer Ser Ser 145 150 155 160 Asn Thr Asp Ser Asn Ser Asn Ser Ala Gly SerSer Thr Ser Gly Gly 165 170 175 Ser Ser Thr Tyr Gly Tyr Ser Ser Asn SerArg Asp Gly Ser Val 180 185 190 20 266 PRT Artificial FRAGMENT FORRESISTIANCE AGAINST DEHYDRATION STRESS 20 Met Arg Gly Ser His His HisHis His His Gly Ser Ala Cys Glu Leu 1 5 10 15 His Arg Gly Gly Gly ArgSer Ser Met Thr Gly Gly Gln Gln Met Gly 20 25 30 Arg Gly Ser Ile Glu GlyArg Ser Ser Thr Gly Ser Ser Ser Asn Thr 35 40 45 Asp Ser Asn Ser Asn SerAla Gly Ser Ser Thr Ser Gly Gly Ser Ser 50 55 60 Thr Tyr Gly Tyr Ser SerAsn Ser Arg Asp Gly Ser Val Ser Ser Thr 65 70 75 80 Gly Ser Ser Ser AsnThr Asp Ser Asn Ser Asn Ser Ala Gly Ser Ser 85 90 95 Thr Ser Gly Gly SerSer Thr Tyr Gly Tyr Ser Ser Asn Ser Arg Asp 100 105 110 Gly Ser Val SerSer Thr Gly Ser Ser Ser Asn Thr Asp Ser Asn Ser 115 120 125 Asn Ser AlaGly Ser Ser Thr Ser Gly Gly Ser Ser Thr Tyr Gly Tyr 130 135 140 Ser SerAsn Ser Arg Asp Gly Ser Val Ser Ser Thr Gly Ser Ser Ser 145 150 155 160Asn Thr Asp Ser Asn Ser Asn Ser Ala Gly Ser Ser Thr Ser Gly Gly 165 170175 Ser Ser Thr Tyr Gly Tyr Ser Ser Asn Ser Arg Asp Gly Ser Val Ser 180185 190 Ser Thr Gly Ser Ser Ser Asn Thr Asp Ser Asn Ser Asn Ser Ala Gly195 200 205 Ser Ser Thr Ser Gly Gly Ser Thr Tyr Gly Tyr Ser Ser Asn SerArg 210 215 220 Asp Gly Ser Val Ser Ser Thr Gly Ser Ser Ser Asn Thr AspSer Asn 225 230 235 240 Ser Asn Ser Ala Gly Ser Ser Thr Ser Gly Gly SerSer Thr Tyr Gly 245 250 255 Tyr Ser Ser Asn Ser Arg Asp Gly Ser Val 260265 21 343 PRT Artificial FRAGMENT FOR RESISTANCE AGAINST DEHYDRATION 21Met Arg Gly Ser His His His His His His Gly Ser Ala Cys Glu Leu 1 5 1015 His Arg Gly Gly Gly Arg Ser Ser Met Thr Gly Gly Gln Gln Met Gly 20 2530 Arg Gly Ser Ile Glu Gly Arg Ser Ser Thr Gly Ser Ser Ser Asn Thr 35 4045 Asp Ser Asn Ser Asn Ser Ala Gly Ser Ser Thr Ser Gly Gly Ser Ser 50 5560 Thr Tyr Gly Tyr Ser Ser Asn Ser Arg Asp Gly Ser Val Ser Ser Thr 65 7075 80 Gly Ser Ser Ser Asn Thr Asp Ser Asn Ser Asn Ser Ala Gly Ser Ser 8590 95 Thr Ser Gly Gly Ser Ser Thr Tyr Gly Tyr Ser Ser Asn Ser Arg Asp100 105 110 Gly Ser Val Ser Ser Thr Gly Ser Ser Ser Asn Thr Asp Ser AsnSer 115 120 125 Asn Ser Ala Gly Ser Ser Thr Ser Gly Gly Ser Ser Thr TyrGly Tyr 130 135 140 Ser Ser Asn Ser Arg Asp Gly Ser Val Ser Ser Thr GlySer Ser Ser 145 150 155 160 Asn Thr Asp Ser Asn Ser Asn Ser Ala Gly SerSer Thr Ser Gly Gly 165 170 175 Ser Ser Thr Tyr Gly Tyr Ser Ser Asn SerArg Asp Gly Ser Val Ser 180 185 190 Ser Thr Gly Ser Ser Ser Asn Thr AspSer Asn Ser Asn Ser Ala Gly 195 200 205 Ser Ser Thr Ser Gly Gly Ser SerThr Tyr Gly Tyr Ser Ser Asn Ser 210 215 220 Arg Asp Gly Ser Val Ser SerThr Gly Ser Ser Ser Asn Thr Asp Ser 225 230 235 240 Asn Ser Asn Ser AlaGly Ser Ser Thr Ser Gly Gly Ser Ser Thr Tyr 245 250 255 Gly Tyr Ser SerAsn Ser Arg Asp Gly Ser Val Ser Ser Thr Gly Ser 260 265 270 Ser Ser AsnThr Asp Ser Asn Ser Asn Ser Ala Gly Ser Ser Thr Ser 275 280 285 Gly GlySer Ser Thr Tyr Gly Tyr Ser Ser Asn Ser Arg Asp Gly Ser 290 295 300 ValSer Ser Thr Gly Ser Ser Ser Asn Thr Asp Ser Asn Ser Asn Ser 305 310 315320 Ala Gly Ser Ser Thr Ser Gly Gly Ser Ser Thr Tyr Gly Tyr Ser Ser 325330 335 Asn Ser Arg Asp Gly Ser Val 340

1. A method for preventing freeze-induced decrease in protein activitycomprising adding a polypeptide comprising the following amino acidsequence (I) to a protein of interest: (SEQ ID NO: 1)Ser-Ser-Thr-Gly-Ser-X1-Ser-X2-Thr-Asp-X3-X4-X5-X6-X7-X8-Gly-Ser-X9-Thr-Ser-Gly-Gly-Ser-Ser-Thr-Tyr-Gly-Tyr-Ser-Ser-X10-X11-X12-X13-Gly-X14-Val (I)

wherein, x1 represents Ser or Thr, x2 represents Asn or Thr, x3represents Aer or Ala, x4 represents Asn or Ser, x5 represents Ser orThr, x6 represents Asn, Asp, or Lys, x7 represents Ser, Asn, or Lys, x8represents Ala, Thr, or Val, x9 represents Ser, or Arg, x10 representsAsn, Ser, Asp, or Arg, x11 represents Ser, Asn, His, or Cys, x12represents Arg or Gly, x13 represents Asp or Gly, and x14 represents Seror Arg:
 2. The method according to claim 1, wherein said amino acidsequence (I) is the following amino acid sequence (II): (SEQ ID NO: 2)Ser-Ser-Thr-Gly-Ser-Ser-Ser-Asn-Thr-Asp-Ser-Asn-Ser-Asn-Ser-Ala-Gly-Ser-Ser-Thr-Ser-Gly-Gly-Ser-Ser-Thr-Tyr-Gly-Tyr-Ser-Ser-Asn-Ser-Arg-Asp-Gly- Ser-Val (II).


3. The method according to claim 1 or 2, wherein said polypeptidecomprises a repetitive sequence in which said amino acid sequence (I) isrepeated at least twice.
 4. The method according to claim 1, 2, or 3,wherein said polypeptide is a chimeric protein.
 5. The method accordingto claim 1, wherein said polypeptide is obtained by a method comprising:providing a recombinant vector comprising a DNA encoding a polypeptidecomprising the amino acid sequence (I), transforming a cell with saidrecombinant vector to obtain a transformant, and culturing saidtransformant and recovering a polypeptide from the resulting cellsand/or culture thereof.
 6. The method according to any one of claims 1to 5, wherein the protein of interest is an enzyme.
 7. Use of apolypeptide comprising the following amino acid sequence (I) to producean agent for preventing freeze-induced decrease in protein activity:(SEQ ID NO: 1) Ser-Ser-Thr-Gly-Ser-X1-Ser-X2-Thr-Asp-X3-X4-X5-X6-X7-X8-Gly-Ser-X9-Thr-Ser-Gly-Gly-Ser-Ser-Thr-Tyr-Gly-Tyr-Ser-Ser-X10-X11-X12-X13-Gly-X14-Val (I)

wherein, X1 represents Ser or Thr, X2 represents Asn or Thr, X3represents Ser or Ala, X4 represents Asn or Ser, X5 represents Ser orThr, X6 represents Asn, Asp, or Lys, X7 represents Ser, Asn, or Lys, X8represents Ala, Thr, or Val, X9 represents Ser, or Arg, X10 representsAsn, Ser, Asp, or Arg, X11 represents Ser, Asn, His, or Cys, X12represents Arg or Gly, X13 represents Asp or Gly, and X14 represents Seror Arg.
 8. The use according to claim 7, wherein said amino acidsequence (I) is the following amino acid sequence (II): (SEQ ID NO: 2)Ser-Ser-Thr-Gly-Ser-Ser-Ser-Asn-Thr-Asp-Ser-Asn-Ser-Asn-Ser-Ala-Gly-Ser-Ser-Thr-Ser-Gly-Gly-Ser-Ser-Thr-Tyr-Gly-Tyr-Ser-Ser-Asn-Ser-Arg-Asp-Gly- Ser-Val (II).


9. The use according to claim 7 or 8, wherein said polypeptide comprisesa repetitive sequence in which said amino acid sequence (I) is repeatedat least twice.
 10. The use according to claim 7, 8, or 9, wherein saidpolypeptide is a chimeric protein.
 11. The use according to claim 7,wherein said polypeptide is obtained by a method comprising providing arecombinant vector comprising a DNA encoding a polypeptide comprisingthe amino acid sequence (I), transforming a cell with said recombinantvector to obtain a transformant, and culturing said transformant andrecovering a polypeptide from the resulting cells and/or culturethereof.
 12. The use according to any one of claims 7 to 11, wherein theprotein to be frozen is an enzyme.